Previous studies showed that GDF11 may reverse age-related heart hypertrophy, improve functions in the nervous system, and increase angiogenesis in aging animals 3,4. During the embryonic stage, GDF11 could inhibit proliferation and promote differentiation of differentiable cells 8. However, the functions of GDF11 in adults, especially its roles in reversing age-related diseases, remain poorly understood. One potential mechanism could involve GDF11 interacting with FOXO family and inhibit hypertrophy 3. Our network analyses also confirm the involvement of the FOXO family in GDF11 functions in our transcription network. Despite this possibility, the mechanism behind the connection between GDF11 and rejuvenation are limited. Through our integrative network analyses, we aim to expand our knowledge of GDF11 and its functions in adulthood.
Based on our bioinformatics analyses of gene co-expression networks and transcription factor networks, we found that GDF11 is involved in apoptosis, DNA repair, telomere maintenance, transcription regulation, cell survival, angiogenesis and regeneration. We also identified potential novel related transcription factors of GDF11, including MYC, SP family, CREB1 and ETS2. Below we discuss in detail the common and novel functions uncovered by our GDF11 network analyses.
Based on human genetic data analysis, apoptosis is the most common function of GDF11 among systems (adipose tissue, nervous system, cardiovascular system, digestive system, skeletal muscle tissue and endocrine system), and through SMAD/CTCF/MYC signaling and JNK/BAD route to promote apoptosis both in the intrinsic and extrinsic caspase apoptosis pathways (Figure 1). Apoptosis plays a significant role in longevity. In stable cells and continuously dividing cells, apoptosis could eliminate presumably dysfunctional cells. Reactivation of apoptosis may be beneficial in clearing cancerous and senescent cells in aging. During the aging process, apoptosis activity decreases, resulting in a decrease of normal homeostatic cell turnover rate 14. Several experiments have been carried out to verify the role of GDF11 in promoting apoptosis. Wang et al. also found GDF11 could induce apoptosis. They added different concentrations of GDF11 to the culture medium of C17.2 neural stem cells. After 72 hours of culture, compared with the control group, different concentrations of GDF11 treatment group (12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml) showed neural stem cell differentiation, and the high concentration of GDF11 treatment showed obvious apoptosis (the apoptosis rates of the four groups of 12.5, 25, 50, 100ng/ml were 2.1%, 9.8%, 13.1%, 17.7%, respectively) 15. Zhang et al. found that exogenous GDF11 can cause apoptosis in myocardial H9C2 cell line, whereas GDF11 knockdown reduces apoptosis 16. Liu et al. also found that the PANC-1 cell line overexpressing GDF11 by lentivirus showed increased apoptosis when analyzed for the role of GDF11 in pancreatic cancer. At the same time, cells treated with RNAi reduced GDF11 showed decreased apoptosis 17. Millet et al. believe that the TGFβ family promotes apoptosis by binding to downstream SMAD2/3 and inhibiting bcl-2, an anti-apoptotic protein 18. And after silencing TGFβ downstream SAMD2/3, apoptosis of stem cell-like cell lines NRP-154 and NRP-152 was reduced 19.
Our results suggest that GDF11 could promote apoptosis both in the intrinsic and extrinsic caspase apoptosis pathways, and provide a few novel specific GDF11 downstream apoptosis pathways as GDF11/SMADs/CTCF/MYC pathway and GDF11/JNK/BAD pathway (Figure 1). The former perfect previously discovered GDF11/SMADs/bcl-2 intrinsic apoptosis signaling with adding intermediate process of CTCF/MYC/p53. The later give us a new research direction of GDF11 involved in apoptosis. The classical pathway of GDF11 is through SMADs which acts by regulating diverse biological effects by partnering with various transcription factors 20. MYC inhibitor CTCF is one of the GDF11/SMADs downstream transcription factor 11 and also appearing in our GDF11 transcription network with MYC. MYC has been recently linked to longevity 21. A possible explanation for this might be through apoptosis as low MYC could activate p53 and induce BCL2-mediated intrinsic apoptosis. Previous studies have concerned p53 is the directly downstream of MYC and low level overexpression of MYC could induce sustained apoptosis 22. So the GDF11-induced apoptosis signaling through SMADs/CTCF/MYC/p53 which suggested from our results is logical. Besides the GDF11/SMADs/CTCF/MYC signaling, our results also indicate GDF11 involved in apoptosis through JNK/BAD pathway and DR. The other two processes are not as specific as GDF11/MYC pathway, more experiments are needed for confirmation. From our discussion we can see the advantage of bioinformatics analysis of pointing out unknown information for future research. And from the above analyses we believe GDF11 could promote apoptosis through intrinsic and extrinsic caspase apoptosis pathways, and benefit age-related diseases as a reasonable result.
From our results, GDF11 is also involved in DNA damage repair and DNA damage checkpoints. In human cells, DNA damage occurs every day due to internal and external environmental reasons such as UV or x-ray. Extensive and cumulative DNA damage will result in cell carcinogenesis, cell death or apoptosis. Along with ageing, the rate of DNA repair decreases and a large amount of DNA damage accumulates. In this case, age-related diseases are prone to occur, and sometimes even cause cancer 23. Another evidence is that several premature senility syndromes have potential DNA repair deficiencies 24. In this study, we saw GDF11 was involved in DNA repair in many tissue systems, such as adipose tissue, nervous system, cardiovascular system, and endocrine system. Among these systems, GDF11 is involved in DNA damage repair checkpoint and single- or double-strand break repair signaling, and suggests that these DNA repair mechanism is related to PI3K/AKT signaling and SP1, the transcription factor associated to GDF11 at transcriptional level. Many studies have shown that the PI3K/AKT signaling pathway is the direct pathway in response to DNA damage, and several PI3K/AKT signaling downstream proteins guide cell cycle checkpoint activation, DNA repair, and activation of apoptosis after unsuccessful repair 25,26. SP1 is a promoter-binding protein and is a downstream molecule of PI3K/AKT. Most genes have multiple SP1 sites in the proximal promoter region. Studies have shown that the consumption of SP1 makes the cells sensitive to DNA damage, decrease the repair rate, and increase the double-strand DNA damage frequency 26. Beishline et al. found that SP1 appeared at DNA damage region 7.5 minutes after DNA damage and persisted at the DNA break site for at least 8 hours, and the consumption of SP1 inhibited the repair of DNA breaks 27. From our results of gene and transcriptional analysis in this study, GDF11 may be involved in the detection and repair of DNA damage through the SMADs/SP1 signaling pathway and the PI3K/AKT signaling pathway. With the successful repair of DNA damage, the organism will maintain stable internal environment. Normal cell proliferation activity and cell function are conducive to the recovery of organ function in aging tissues.
Our results also link GDF11 with telomere function. Telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, so in each replication the end of the chromosome is shortened, which has been associated with aging. The length of telomere has become a symbol of longevity and telomere shortening is associated with age-related diseases, prolonging telomere length has become the research direction of delaying aging. Our research suggested that GDF11 can up-regulate telomere by regulating telomerase reverse transcriptase (TERT), through transcription-related factors SP1, ETS2 and GDF11/SMAD/CTCF/MYC signaling pathways. Telomerase acts as reverse transcriptase in the elongation of telomeres. TERT is a catalytic subunit of the enzyme telomerase, which, together with the telomerase RNA component (TERC), comprises the most important unit of the telomerase complex. The TERT gene promoter region has all SP1, ETS2 and MYC binding sites, and the three could synergistically activate TERT transcription and direct telomere elongation 28. Among them, SP1 is effective but not necessary. SP1 overexpression can activate TERT, but SP1 site mutation has little effect on TERT 29. ETS2 is important for driving TERT gene expression, silencing of ETS2 results in a decrease in TERT gene expression 30. MYC plays an important role in TERT expression and telomere elongation. MYC regulates TERT in dual-direction, and there is a feedforward regulation 31. MYC can activate telomerase to extend the terminal telomere of the gene and return the cell to a sustained division state32. SP1, ETS2 and MYC all belong to GDF11 transcriptomic network from our results. And transcription of TERT can be inhibited by E2F transcription factor 1 (E2F 1)33, which is also involved in the GDF11-related transcription factor regulatory network. These results strongly suggest that GDF11 may have bidirectional regulation of TERT and telomere length. It can promote telomere synthesis by up-regulating TERT through SP1, ETS2 and MYC. At the same time, TERT and telomere synthesis can be inhibited by MYC and E2F1, also TERT level can be precisely regulated by MYC feedforward reaction. These bidirectional regulation for telomerase is necessary. Because TERT overexpression will induce tumor-like hyperproliferation, which is unfavorable to tissue and organ stability. But with TERT transcription bidirectional regulation, telomere can be regulated by controlling level and prolong telomere length, slow down cell senescence, restore organ function, maintain body homeostasis and prolong life of organism to a certain extent. Recent study has shown that GDF11 has a positive effect on the maintenance of telomere length which supports the conclusions of this study, but that paper does not provide specific mechanism analysis 34. In this study, bioinformatics analyses showed that GDF11 could through SMAD/CTCF/MYC signaling pathway, SP1 and ETS2 regulate TERT to maintain telomere length, which provides direction and target for subsequent experimental verification.